Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done to investigate how the secondary and tertiary structures of proteins are formed and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions, such as: How do specific and nonspecific interactions with the surroundings determine the folding pathways, the roughness of the energy landscape, and the thermally and kinetically accessible conformational substrates? On what range of timescales do particular conformational and folding events occur in crowded environments and/or membranes? Addressing these questions presents the need for further studies with time-resolved spectroscopic techniques that can provide the necessary time resolution and structural sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformational probes that can be used to generate detailed structural interpretations of the transient folding species and their dynamics over a wide range of timescales. A comprehensive set of experiments are planned to gain new insight into the understanding of various aspects of the protein folding problem, as well as the structure-dynamics-function relationship of membrane proteins. The technical goals are to: (a) develop new spectroscopic probes and methods for protein folding and binding studies;(b) investigate how macromolecular crowding and confinement affect the kinetics of protein folding and aggregation;(c) study the early kinetic events in membrane protein folding;(e) probe the conformational dynamics underlying membrane protein function. Achieving these specific aims should result in not only new experimental methods for various biological applications, but also new findings that would help improve our understanding of many fundamental issues in protein folding, thus making practical and direct contributions to the field. PUBLIC HEALTH RELEVANCE: Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Achieving these specific aims should result in not only new experimental methods for folding and binding studies, but also new insights into the understanding of many fundamental issues in protein folding, thus making practical and direct contributions to the field and having fundamental importance for public health.